Integrated nucleic acid test system, instrument and method

ABSTRACT

A method of analysing nucleic acid using apparatus comprising a reaction chamber and plurality of sensors located in the base of the chamber, with each sensor preferably located within a respective well. The method comprises flowing a fluid containing the nucleic acid or fragments thereof into the reaction chamber. While the chamber is fully or at least partially sealed, amplification of the nucleic acid or said fragments is performed within the chamber using an amplification primer or primers whilst detecting the generation of amplicons using said sensors. Sequencing or hybridisation is then performed on the amplicons, and sequencing or hybridisation is detected using said sensors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/306,554, filed on Oct. 25, 2016; which is a national stageentry of International Application No. PCT/EP2015/059097, filed on Apr.27, 2015, which claims priority of United Kingdom Patent Application No.1407334.0, filed on Apr. 25, 2014, which is entirely incorporated intothe present application by reference.

TECHNICAL FIELD

The present invention relates to a system, instrument and method forprocessing nucleic acid. The invention facilitates control and operationof the workflow and components of the instrument/system. The processingmay involve, by way of example, DNA/RNA sequencing and/or performing animmunoassay.

BACKGROUND

There have been significant breakthroughs in the development ofbiological assays. Such assays include immunoassays, DNA sequencing,analytical chemistry, and coulometry. Some of these assays have beendeveloped as instruments for diagnostics. Many of these assays are quitecomplex and require specialised knowledge to operate and interpret.

Within the field of genetics there are applications for whole genomesequencing, de novo sequencing, gene expression analysis, and detectingsingle nucleotide polymorphisms (SNP). Each of these tests presentdifferent levels of information and complexity of testing. In certaincases more than one of these tests will be required and it is up to theclinician to decide how to progress.

Most test workflows have three major aspects: sample preparation,detection, and interpretation of the results. The sequencing bysynthesis technique for genetics is an example of a complex processhaving many steps for each of these aspects.

The preparation phase may start with extraction of template nucleic acidfrom the sample provided, followed by purification, fragmentation of thenucleic acid into workable lengths, clonal amplification of the templatefragments, hybridisation/ligation to a primer/probe, andligation/immobilisation to a surface or bead. The sequencing bysynthesis step itself requires flowing different nucleotide types(dNTPs) across the template colonies, wherein nucleotides becomeincorporated with the template nucleic acid.

The detection phase may employ various technologies such aselectrophoresis, chemical luminescence and optical cameras, measuringelectrode impedance/capacitance change, measuring immobilised DNAintrinsic charge change, or monitoring proton concentration with an IonSensitive Field Effect Transistor (ISFET).

The Interpretation phase is the analysis and extraction of informationfrom the detection results. For example, this may include: determiningthe sequence of nucleotide incorporations detected from each fragment;determining their alignment/reassembly to form the whole genome; ortheir comparison with known sequences. The results may guide treatmentor suggestion of complementary products, although the final result maybe days after the initial sample collection.

Of the detection processes identified above, one of the most excitinginvolves the use of ISFETs and other chemical FETs (ChemFETs). The useof ISFETs to sequence DNA and DNA fragments (as well as RNA and RNAfragments) is described for example in WO03/073088. This work hasdemonstrated that the incorporation of nucleotides (A,T,C,G) duringextension of a DNA strand can be monitored by using an ISFET to measurethe variation in ionic concentration as a by-product of the reaction.When a nucleotide extends a DNA strand, it releases pyrophosphate whichis hydrolysed and generates H+ ions, reducing the pH. In a similar way,an ISFET can be used to detect hybridisation whereby a hybridisationprobe attaches to a matching sequence on a DNA strand.

An extension of this approach using very large scale FET arrays isdescribed in US2009/0026082 and provides for massively parallelanalysis. By analysing a large number of DNA fragments in parallel, andthen aligning and “stitching” together the results, long sections of DNAmay be sequenced in a relatively short time.

In the case of an ISFET based assay, the ISFET chip will be designed andmanufactured to perform one or a set of predefined assays. For example,The ISFET chip will be configured to detect the presence of a set ofSNPs in a sequence of DNA. The sample is first prepared on thelaboratory bench. This may involve enriching the sample to removematerial other than the cells, lysing the enriched cells to release theDNA, and performing amplification on one or more DNA sequences. Duringor following this process, the amplified DNA sequences are attached tomicro beads. These beads are then introduced to the chip. This mightinvolve depositing the beads into wells formed above the individualISFETs, e.g. using magnetic beads to introduce one bead into each well.The chip can then be inserted into an instrument where the sequencing isperformed. The instrument causes different nucleotides (A,T,C,G) to beflowed cyclically through the chip, with a washing step between eachnucleotide flow. Electrical signals representing chain extensions aredetected. The result provided by the instrument is a chain extensionsequence for each ISFET. This data is then analysed, for example using adesktop PC connected to the analyser, to weigh the results according totheir prevalence. Sequences that have a high prevalence will be recordedas valid sequences, whilst sequences with a relatively low prevalencewill be recorded as being due to noise. The valid sequences may then beused to determine the presence or absence of a SNP(s) in the analysedsample. Of course other workflows and analysis routines are possible.

In addition to these essentially laboratory-bound assay processes,point-of-care assay procedures and systems have been developed. Forexample, DNA Electronics (London, UK) has developed a genetic testingkit that allows procedures such as that described above to be conductedby essentially unskilled persons at a point-of-care or point-of-sale. Inmany cases, testing using an ISFET-based assay will form only one partin a workflow that will be followed by a skilled technician. Based onthe result of the ISFET-based assay, decisions may need to be madeconcerning further tests, and these tests will need to be performed,e.g. using further ISFET-based assays.

WO2011/034790 and US2012/0109531 present exemplary biological andphysiological assay schemes and instruments that provide a degree ofautomation and flexibility. They are not however directly applicable tothe handling of nucleic acids.

SUMMARY OF THE INVENTION

The inventors have envisaged a system able to flexibly run assays on anucleic acid sample. Depending on the program that is run, differentfunctions may be performed. These may be based on the data fed-back tooptimise the instrument results.

In accordance with a first aspect of the present invention there is amethod of analysing nucleic acid using apparatus comprising a reactionchamber and plurality of wells located in the base of the chamber, andat least one sensor located within each well. The method comprisesflowing a fluid containing the nucleic acid or fragments thereof intothe reaction chamber. While the chamber is fully or at least partiallysealed, amplification of the nucleic acid or said fragments is performedwithin the chamber using an amplification primer or primers whilst thegeneration of amplicons may be detected using said sensors. Sequencingor hybridisation is then performed on the amplicons whilst detecting thesequencing or hybridisation using said sensors.

Other aspects of the invention are set out in the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an exemplary workflow carried out using a systemcomprising an array of test elements;

FIG. 2 illustrates a module of an ISFET-based chip suitable for use inimplementing the workflow of FIG. 1 ;

FIG. 3 illustrates schematically various biological operations that mayform part of the workflow of FIG. 1 ;

FIG. 4 illustrates schematically an instrument for implementing aworkflow such as that illustrated in FIG. 1 ;

FIG. 5 is a block diagram of conceptual elements of the instrument ofFIG. 4 ;

FIG. 6 illustrates schematically hardware and software components forimplementing an Operating System;

FIG. 7 illustrates schematically components of a “biological” operatingsystem and an application layer for implementing biological workflows.

FIG. 8 is a flow diagram illustrating a process for analysing nucleicacid or fragments thereof; and

FIG. 9 illustrates schematically a cartridge suitable for implementingthe method of FIG. 7 .

DESCRIPTION

A system and process are proposed, by way of example, that allow anintegrated array of test elements (or “cells”) to be configured toperform various assays and even reconfigured during the performance ofan assay in dependence on intermediate results. In other words,intermediate decision points are introduced into the workflow followedby the assay, and the decisions made at each of the decision pointsdetermines how the workflow proceeds from that point, with the testelements being configured accordingly. Such configuring may involvecontrolling the flow of fluids into the test elements, for example tointroduce appropriate primers and hybridisation probes and/or activatealready deposited primers or hybridisation probes.

In a typical system, a chip comprises an integrated array of testelements, for example ISFETs, with different groups of test elementsbeing arranged to perform different tasks. For example, a first group ofISFETs may be arranged to perform hybridisation to measure thesimilarity of a sequence of the DNA being analysed to a hybridisationprobe bound within the test area. Further groups of ISFETs may bearranged to perform sequencing on respective different sections of theDNA under analysis. A decision point incorporated into the workflowmakes a decision regarding which section of DNA to sequence based uponthe result of the genotyping process.

In an exemplary situation, a patient admitted to a hospital showssymptoms that could indicate trauma, heart attack, local infection orsepsis. The doctor must determine which of these is true and administerthe appropriate treatment. Referring to FIG. 1 , the horizontal axisrepresents an exemplary timeline for the resulting workflow on aparticular sample. A blood sample is introduced to a sample ingress portof the test instrument incorporating an ISFET-based assay chip. Theinstrument may comprise pumps and valves, in conjunction withmicrofluidic flow channels and fluid gates, for directing the receivedsample to appropriate parts of the chip.

As a first phase of the workflow, diagnosis is made between conditionssuch as sepsis, local infection, trauma or Acute Myocardial Infarction(AMI). The result in this example is a diagnosis of sepsis.

It is now desirable to identify the family of the pathogen (to identifysepsis species and resistance genes) responsible for the infection. Thisis carried out by applying the extracted DNA to a set of ten PCR panels.These are selected as a result of the sepsis diagnosis. In someembodiments, all of the available modules are integrated onto a singlechip whilst, in others, modules are provided on different chips. In anycase, each of the ten PCR panels of the selected module is configured toperform PCR on a different targeted DNA sequence corresponding to adifferent family of pathogens. Each PCR Panel may comprise one or morewells each sitting above an ISFET sensor. An amplification primer may beimmobilised at the base of each well or in solution, with differentamplification primers for each Panel. The fluid control system isoperated to cause a fluid containing all four nucleotides (A,T,C,G) toflow through each of the Panels. A heating and temperature sensingelement is incorporated into the system to allow PCR amplification to beperformed. In the example illustrated, a positive result is provided byPanel 3 (i.e. amplicons are generated—above some threshold level—only byPanel 3) thereby identifying the family of pathogens. In an alternativearrangement, rather than being immobilised within the wells, theamplification primers may be introduced into the panels via the fluidcontrol system, e.g. together with the nucleotides.

In an alternative architecture, rather than having a plurality ofmodules available to perform different sets of PCR assays and selectingone of these based upon the triage diagnosis, a generic module may beprovided with a set of panels that are “programmed” by flowing specificamplification primers through these, resulting in the immobilisation ofthe desired primers within the panels (or rather within the wells of thepanels).

The third phase of the workflow is to identify the particular species ofpathogen from within the identified family. In this embodiment thismakes use of a set of hybridisation tests performed by six differentmodules, although this could alternatively be implemented by detectionof amplification or sequencing, and the hybridisation phase may beomitted. Each module comprises one or more wells with each well sittingabove an ISFET. A hybridisation probe is immobilised at the base of eachwell, with different probes for different modules. The sample, afterpreparation, is transported to the modules by appropriate operation ofthe fluid control system. A heating and temperature sensing element isincorporated into the system to allow hybridisation cycles to beperformed repeatedly. In this example, a positive result is generatedonly by the Species 2 test. In an alternative arrangement, thehybridisation probes may be transported into the modules using the fluidcontrol system.

As with the PCR modules, hybridisation modules may be selected from agreater number of available modules, or may be obtained by programming aset of generic modules so as to immobilise appropriate hybridisationprimers in the appropriate wells, or may be pre-programmed.

Considering further the second (amplification/PCR) phase and the third(hybridisation) phase, as described, these may be performed by separateamplification and hybridisation modules. An alternative approach howeveris to perform these phases within the same modules. This might beachieved for example by performing and detecting amplification within agiven module and then activating or utilizing hybridisation primersalready immobilised within the module to allow hybridisation to becarried out and detected. The activation process may involve heating andremoving a layer of wax within the wells of the module to exposeunderlying hybridisation primers, or the hybridisation may be activatedby adjusting the temperature to facilitate hybridisation, or thehybridisation may occur during the amplification phase.

The fourth and final phase of the workflow is optionally to sequence therelevant section(s) of the DNA, again using an appropriate module of thechip. Sequencing first requires that the DNA segment(s) to be sequencedhave been amplified. Amplification can be performed in a pre-chamber influid communication with the sequencing chambers, e.g. using PCR.Alternatively, amplification may be performed in the sequencing chambersthemselves. Amplification proceeds as described above with respect tothe second phase of the workflow. The sequencing chambers contain one ormore wells, above respective ISFETs, with a sequencing primer beingimmobilised at the base of the wells. Again, the fluid control system isoperated to cause the prepared blood sample to flow into the module.Nucleotides (A,T,C,G) are then flowed through the module in sequence.Sequencing of different segments of the DNA may be performed inparallel, with the result data being aligned and stitched together.

In some embodiments, sequencing may be performed within the same modulesthat are used to perform the phase 2 amplification and the phase 3hybridisation.

The detected sequence(s) can then be added to a database and comparedwith known pathogens to determine if the pathogen is known or unknown. Arecommendation for treatment may be given upon final completion, whichcould include use of a drug with a pharmaco-genetic relationship. Anoption may be available to perform genotyping tests for the tailoreddrug.

Identification of the sample at each phase leads to another assay havinga plurality of further possible identities and so on. After each phasein the workflow, subsequent workflow decisions are made. Also theintermediate results can be used to inform treatment decisions. Forexample, if the result of the first phase indicates sepsis, preliminarytreatment for that may begin. The second phase assay identifies whatform of sepsis is present (viral, bacterial, fungal, etc). If the secondphase identifies a bacterial presence, treatment with a broad spectrumantibiotic may be recommended. As the workflow progresses, furtherintermediate treatment decisions can be made.

Furthermore, it will be appreciated that, during a particular assaystage, decisions about the assay may be made. For example, duringsequencing of a nucleic acid at the fourth phase, each baseidentification may be used to determine whether to continue sequencingfor further identification, stop sequencing, or flow a particular orderof nucleotides.

In the case of modules that perform amplification, by monitoring allreaction volumes it is possible to switch off or ignore any downstreamprocesses for any chambers that show no amplification. For thosechambers that do show amplification, a decision is needed on whether ornot to proceed to the next stage of the process. For example, if aresistance gene is identified, this might provide enough information tomake a recommendation to the physician to tailor the treatmentspecifically for this resistance gene and therefore the workflow forthat sample might end at this stage. If, however, the resultingamplification is still ambiguous or requires further analysis, adecision can be made to progress to hybridisation and sequencing.Similarly to the amplification mode, monitoring these modes allowsdecisions to be made to progress or not, to continue to monitordownstream events or not, including stopping the test altogether andmaking a recommendation for treatment.

In the cases of either trauma or AMI being diagnosed, the opportunityexists to screen the patient for drug interaction information, forexample establishing the genotype for drugs such as warfarin or PLAVIX.In both of these cases, the genotyping can be done on the samplecollected during the triage (which will contain large amounts of thepatient's cells and DNA). Genotyping can be determined using the firstmodality of the system—PCR or isothermal amplification and detection.There is not likely to be a need to progress beyond this stage.

FIG. 2 illustrates a module of an ISFET-based chip suitable for use inthe process described above. This module, at least in terms of itsphysical structure, might be generic to all of the different assay typesperformed by an instrument (e.g. as exemplified by the above discussionwith reference to FIG. 1 ), e.g. amplification, hybridisation andsequencing. In some cases the module may be configured to perform two ormore of amplification, sequencing and hybridisation. To the left of theFigure there is illustrated a “pre-amp” stage that is configured toprepare a sample for subsequent genetic analysis. This stage may, forexample, isolate cells in a blood sample and extract the DNA from thecells. The pre-amp stage then supplies the enriched sample to a reactionchamber comprising five wells, each exposed to ISFET sensors. The inseton the right hand side of the Figure illustrates a side, cross-sectionalview through one of the row of sensors. Each of the reaction chamberscomprises ingress and egress fluid ports, and a central space forholding a volume of fluid. The ISFET sensors are arranged on the base ofthe wells. Additionally, the module may comprise a heater andtemperature sensor for heating fluid in the chambers.

Depending upon the assays to be performed, amplification and sequencingprimers, and hybridisation probes, may be immobilised within the wellsof the module. As a particular example, one might consider the casewhere the module is configured to perform amplification (PCR) followedby sequencing. In this case, the prepared sample, containing extractedand fragmented DNA, may be flowed into the chambers of the moduletogether with an amplification primer and nucleotides. The temperatureis then cycled to perform PCR amplification. Sequencing primers areimmobilised within each well. Sequencing is then performed on theamplicons and the results read out from the ISFETs.

One approach to achieve multi-mode workflows is to have each modeassigned to a specific chip, and to assemble the chips into a cartridgesuch that the cartridge has all of the necessary modes but withouthaving the ability or requirement to have each chip capable of switchingbetween modes. This design would overcome anticipated challenges such asthermal compatibility and functionalization, but would bring with it amore complex design for the fluidic handling and control, etc.

In an alternative approach, a portfolio of test cartridges areavailable, each with specific functions or target diseases, and thesecan be selected dependent upon the results of earlier steps. Selectionof the downstream tests (cartridges) could be an automated process orone involving a user-intervention (to plug two cartridges together, forexample). Some examples, all using the starting point of a triage systemwith sample enrichment, are as follows:

-   -   Trauma: On diagnosis of trauma, the appropriate downstream test        may be to measure the pharmacogenomics profile of the patient        for drug interaction information. The triage cartridge, used to        make the diagnosis of trauma, effectively becomes the sample        prep module and can be plugged/slotted into the appropriate        trauma cartridge for the subsequent genotyping reactions to be        executed.    -   Sepsis: the appropriate downstream test may be to identify the        specific species or strain, and also identify any resistance        genes that exist in the individual. The enriched sample from the        triage component can be plugged into the sepsis cartridge and        downstream analysis follows. There is the possibility that the        prescribed treatment has a genetic factor to consider, and there        is the option to genotype the individual. Again, either the        triage sample could be used to run genotyping tests (or these        could be standalone rapid tests).    -   Local infection: the appropriate test would be determined using        other information provided, including physician observations, to        select the appropriate panel of tests. Because the site of        infection and/or the cause of the insult will influence the        tests to be performed, it is reasonable to picture a number of        different cartridges being available (as opposed to having one        cartridge to cover all potential infections within a hospital        environment). To make the system optimal, there could be a need        to plug in the relevant test cartridge, which will be influenced        by several factors and data.

Considering now FIG. 3 , this illustrates an example process foranalysing a sample of DNA. It is assumed that functionality has beenassigned to a chip by depositing specific primers to known regions/wellswithin the chip. Given that it is known a priori what has been depositedand where, it can be determined what has been amplified based upon thesequences used for the primers. In this example, the primers used in theinitial mode of amplification get modified with the correspondingsequence from the sample and ultimately are used to generate the productto be sequenced later in the workflow.

A sample enters the reaction chamber, amplification takes place (withreal time detection) and the printed primers are modified to reflect thetarget sequence. After the amplification mode has completed,solution-phase primers are flushed away and replaced by sequencingprimers. Being able to detect the hybridisation event might provideadditional sequence information. Sequencing can then follow by flowingone or more nucleotides across the reaction chamber.

This system will require a less sophisticated fluid handling and controlsystem compared to other methods, but may require additional mechanicalactuations to allow different modes to follow each other.

In principle, the system has the ability to perform the amplificationmode, detect any amplification that takes place, and can decide whetheror not to proceed to the next stage. In instances where there is noamplification, the system can determine not to monitor any downstreamreactions, or to stop any further reactions taking place. Similarly forthe subsequent modes, failure to detect hybridisation can switch off thedownstream sequencing mode whereas detection of hybridisation can allowa decision to be made to progress or not.

An instrument comprising or for use with the chips/modules describedabove comprises an operating system for interfacing with the componentsof the instrument and is arranged to receive data to configure orreconfigure the instrument. In certain embodiments, the instrument isarranged to receive real time data representing biological data aboutthe sample, and the operating system can reconfigure the workflow of atest on the sample or reprogram components of the instrument. In thissense there is feedback of the test data to the instrument to operatethe instrument in an optimal way. In certain embodiments, the instrumentis arranged to receive data from an external source or an applicationrunning on the operating system and the operating system can configurethe instrument to run biological tests in a customised way.

FIG. 4 illustrates at a very high level an instrument including a chipas described above, including both an operating system and anapplication layer (comprising one or more applications). The instrumentreceives input, which may be automated or manual input, from a varietyof sources. The Figure suggests three sources: An application medicaldatabase, hospital rules, and clinician generated input. The instrumentprovides as an output a diagnosis and/or a recommended treatment regime.

The physical components of a biological instrument will depend on theassay type and technology employed. FIG. 5 is a block diagram ofconceptual elements of an instrument according to a preferredembodiment.

-   -   A sample preparation device;    -   A cartridge comprises the means to combine the sample and        reagents for a bio-chemical reaction to create complexes to be        detected;    -   A microfluidic network ensures that the sample and reagents are        directed from component to component in a controlled way;    -   Reagents are stored within the instrument connected to piping        and pumps, where valves and solenoids control the flow of the        reagents;    -   A sensor or sensor array proximate or integrated with the        cartridge detects signals from or properties of the complex.    -   An interface circuit is coupled to the sensor(s) to read out the        electrical signals from the sensors    -   A control board comprises    -   A signal processors    -   ACPU/controller    -   A memory    -   An OS    -   An application layer

While certain embodiments of the OS may be used with a moderatelycomplex assay such as an ELISA, preferred embodiments are used withcomplex instruments with longer, flexible workflows and which providegreater depth of data. Complex instruments and assays, such as geneticsequencers, provide a large data stream which can be fed back to the OSand offer a number of decision points to change the workflow or selectdifferent assays.

In an exemplary instrument:

-   -   the assay is DNA sequencing;    -   the sensor cartridge is a semiconductor chip having a massive        array of ISFETs;    -   the sample preparation comprises emulsion PCR on the template        and binding to separate beads provided to wells proximate the        ISFETs;    -   the reagents dATP, dTTP, dCTP, dGTP and wash are provided by a        pump to the wells;    -   the ISFET signal is read one row at a time to a multiplexed        array of analogue to digital converters (ADC);    -   the signal processor filters and determines nucleotide        incorporation events, which are stored in a massive memory.

The Operating System controls these components and workflow.

Preferred embodiments of the instrument may be designed to accept aparticular biological sample from a patient such as: blood, saliva,stool, mucus, etc.

The Operating System provides an interface between the instrumenthardware and an application layer which contains a set of instructionsto run the instrument. The OS may be considered similar to a combinationof the BIOS (Basic Input Output System) and the conventional OperatingSystem of a personal computer. The OS has access to and control of thecomponents of the instrument such that it can turn them on/off or setproperties of them. The OS defines how instructions affect theinstrument and is responsible for translating the high levelinstructions into low level control of the components. The OS may alsobe designed to safeguard the instrument, for example by ensuring thatthe order of instructions is logical or parameters requested are withindesign limits.

The OS comprises hardware and software aspects. The hardware maycomprise a control board having a processor, a memory, a data bus, andcomponent drivers. The control board may be electrically connected tofurther hardware elements of the instrument such as a sensing chip, orit may share the board with such further hardware elements. The softwareaspect is encoded into non-volatile memory, preferably on the controlboard, and determines the operation of the OS hardware. Whilst variousOS systems may be designed using well known architecture for receivingand processing signals and instructions to control instrumentation, anexemplary embodiment is illustrated in FIG. 6 and described below.

A non-volatile memory programmed with the OS code forms the OS firmware30. When this code is read by the CPU 31, the control board is able tooperate the instrument. One or more applications may be stored on amemory 32 to provide machine instructions for controlling the instrumentin a customised way. The CPU reads code from the application memory 32which is interpreted by the OS to create instructions for controllingthe instrument. The CPU receives data from external sources on data bus36. The data may be sensor data about the biological sample, data aboutthe instrument hardware, and control feedback data. The data may bedigital data or analogue data in which case the control chip will alsocomprise analogue to digital converters. The data may be stored into adata memory 33. The processor 38 is coupled to a plurality of drivers 34which provide signals to operate components. For example, the driversmay open or close a reagent valve (digital control) or control thetemperature of a heater (analogue or PWM control). The control board mayalso comprise a transceiver and data port for communicating withexternal devices.

In computer science terms, the OS may include Dynamically LinkedLibraries (DLLs) which provide code blocks which may be called fromapplications. They are a shared and reusable resource. The advantage ofDLLs are the provision of commonly used code, saving reprogramming byeach application and ensuring that certain functionalities are robust.For example, in a genetic instrument, a DLL may implement PCR of asample on a cartridge. In pseudo-code the application might simplyinstruct:

-   -   PCR (number_cycles)

Which the OS implements with a set of code that turn on heaters to setthe temperature of the cartridge to a denaturing temperature,hybridisation temperature, and extension temperature with suitabledelays between steps and repeats this cycle based on the parameter‘number_cycles’. The application is saved from re-writing this code andthe temperatures will not exceed design limits.

Equivalent to the control board of FIG. 6 , a single large integratedsemiconductor chip may be fabricated incorporating memory, processor,drivers and data input/output connections.

Alternatively, the instrument may have a data port coupled to theinstrument components and arranged to receive control signals and senddata through the data port to a remote computer. The remote computerwould comprise a processor and OS and be arranged to receive data fromthe instrument, run applications, and send controls signals to theinstrument. In this scenario, the instrument system would comprise theinstrument components and the remote computer.

Conceptually the instructions may be provided to the OS by a separateapplication layer or held in a memory part of the control chip. Theinvention is not intended to be limited by the architecture whichprovides the instructions to the OS.

The approach presented here considers different levels at which thesystem may be configured and/or reconfigured between the action of auser requesting a test and the generation of test results. Depending onthe test requested and the data received, the operating system may makechanges to the workflow or operation of hardware components. Theinstrument is thus able to optimise each test and it is conceivable thatno two tests will be run the same.

If a single instrument is termed a system, then a group of instrumentsor instrument and peripheral hardware may be called a super-system.Components of the instrument may be called sub-systems. Examples ofperipheral hardware include sample preparation devices, bioinformaticscomputers, etc. Examples of instrument components include reagentdelivery units, sensors, cartridges, etc.

At the super-system level, a combination of instruments and/orperipheral devices are connectable such that the sample may be arrangedto flow to a plurality of the instruments or peripheral devices forprocessing. For example, the OS may receive data about the sample type(blood, saliva, etc) and choose a particular sample preparation deviceand run conditions. In another example, the OS may decide to test asample on multiple instruments in parallel to obtain a faster, morecomplete result.

At the system level, the OS may enter a mode of testing or a select aparticular type of test cartridge, (selecting a sepsis/onco chip, withroutes for phase I or phase II testing).

At the sub-system level, circuits on the chip may be configured toswitch sensors, heaters, noise filters, and data acquisition elements onor off.

Further examples of re-configurability are provided below.

At the Super-System Level, the OS receives past test data and determinesthat there is an endemic Hospital Acquired Infection and arranges forall instruments to perform screening on all samples using infectiousdisease assays. The OS would select these assays in addition to theassay selected by the user. The OS might change the workflow, normallyrequiring determination of the infection variant for subsequent tests,by assuming that all patients have the same local disease and stoppingdeep DNA sequencing once the OS has confirmed the presence of theinfection. The OS could then communicate with an external device toreport the endemic infection to the hospital or government healthagencies.

At the Sensor Chip level, data processing could be improved by modifyingor selecting different circuit parameters based on initial sensor signalquality that suggest high background noise. The OS could redirect theraw sensor signal through a filter that rejects certain noise to allowreal time signal improvement rather than need to discard the whole dataset at a later date.

At a GSIC level, the OS could direct one or more GSICs to amplify DNA intheir reaction well. Each GSIC would set the heater temperature,reference electrode voltage, sensor voltage and accept reagents asneeded to comply. If the OS receives sensor data indicating poor copycount in a particular well, it could reinstruct the respective GSIC tofurther amplify the DNA in that well.

The OS control of the instrument components may be at a simple orcomplex level. For example at a high level the OS could simply instructa component to perform a function or achieve a setting, wherein localcircuitry would ensure compliance. At a complex level the OS couldconstantly monitor and control a component to achieve a function orsetting.

To provide reconfiguration of the instrument components or workflowduring testing, the OS is arranged to receive real-time data from theinstrument. The data may be sensor data about the biological sample. Thedata is real-time in the sense of being generated by sensors during thetest run, and not limited to data that is produced at a given instant intime.

The CPU may have dedicated ports to capture events or important signals.This enables the OS to be event driven, making decisions aboutreconfiguring the instrument as and when events occur. Alternatively theCPU may fetch data from memory 33 at points determined by the OS orapplication, such as when a decision point is reached in the workflow.The latter is appropriate where there is a large stream of data and theOS determines the best time to make decisions. For example, with agenetic assay, the OS may wait until an event is received indicatingthat gene-specific amplification has finished before deciding whether todo sequencing. The OS may then periodically recall data from memoryabout the sequences of particular wells to decide when to stopsequencing.

An application layer may be provided to interface with the OS layer. Oneor more applications may be loaded onto a memory to provide machineinstructions to the OS to operate the instrument in a specific way. Anapplication is a set of machine instructions readable by an OperatingSystem. The application may be preinstalled with the instrument orloaded by a user. The program may be customised for a particular user(such as a hospital) to run a specific genetic test (such as detectinfectious disease from a blood sample) according to a specific workflow(such as repetition of one process or skipping of another process).

In preferred embodiments, the application is responsible for configuringor reconfiguring the instrument hardware and workflow. For example, anapplication installed by a particular hospital might instruct deepersequencing of a sample if it detects that a certain virus has beendetected. The hospital might have a policy to check all samples for thisparticular viral strain that is now endemic to a ward such that futuretests become reconfigured in addition to what the user requested.

The application may provide the interface for the user to theinstrument. Different applications may provide different options totheir users, from a single start button to a complex array of optionsproviding flexibility. The OS interprets the applications' instructionsbut maintains overall control and responsibility of the instrument. Forexample the program may instruct the flowing of a reagent but the OS hasmore direct control of the pumps and valves and may override a requestto pump a reagent once it has been consumed.

In a further embodiment, the application is loaded onto and operablefrom a computer external to the instrument. This remote computer may bea server, tablet, cloud computer, or smartphone, and maybe connectableto other computers. The instrument is connectable to this remotecomputer, preferably via a port on the control board. This connectionallows the remote computer to receive biological test data from theinstrument and send instructions to the OS. The remote computer maycontain or be able to access a database of medical data about thepatient providing the sample. This remote application may implementcase-base-reasoning, heuristics for medical decision making, or machinelearning such that the application can use the medical data to makeintelligent decisions about how to configure the instrument. This mayinclude instructing the instrument OS to use a particular workflow, setcomponent settings, and/or run a particular assay. The remoteapplication may receive real time data from the OS to revise itsdecisions and reconfigure the instrument.

For example, a doctor using the remote application requests testing fora patient. The remote application recalls the patient's medical historyand physiological results and compares these to similar medical cases torecommend a genetic test to detect the presence of known cancerbiomarkers. The application instructs the OS to load an oncology assaycartridge. The application can optimise the sequencing workflow knowingwhich bases are predominant for these genes. Having received real-timedata from the instrument about certain genes, the application instructsthe instrument to stop further sequencing. The application theninstructs the instrument to load an assay to test for the patient'sresponse to certain treatments. The application receives the finalgenetic results, adds them to the medical database, and outputs atherapeutic recommendation to the doctor. The application continues tobuild correlations between the genetic results and the medical databaseto update its case-based-reasoning, such that future similar cases maybe tested in a more refined way on the instrument.

To enable the instrument to be particularly flexible and customisable,the instrument is arranged to accept and install one or moreapplications into a memory. These applications may be written by thirdparties desiring to optimise the instrument operation or implementparticular protocols. This allows for third parties to experiment withand optimise the instrument's workflow and components. Alternativelycertain users may have specific needs, provide a certain preferredsample type, or need to implement specific medical protocols or medicalinsurance policies. The application provides instructions to configurethe instrument before testing starts and/or reconfigure the instrumentupon receiving real-time data from the OS.

For example, a particular hospital only uses blood samples and desiresto test each sample for a E. coli in addition to any test requested, andachieve a 90% confidence interval for certain biomarkers. Theapplication instructs the instrument to purify the sample in aparticular way optimised to capture patient and microbial components andthen run both a bacteria assay cartridge and human genotype cartridge.Only a simple presence/absence test is requested for the bacteriacartridge whereas deeper sequencing is requested for the human genotypecartridge. The OS receives and sends real-time data to the application,which requests the OS to further sequence the DNA to determineantibiotics resistance if particular bacteria is present and the patientdata indicates adversity to antibiotics, or to rerun a test if eitherresult is identified by the OS as less than 90% reliable.

The OS is designed to receive “machine code” and translate these intooutput signals to control the assay. Machine code is usually a binarystring compiled from code written in a high level programming languagesuch as C++. Depending on the implementation of the OS, the machine codemight provide simplified, high level instructions or more customisablelow level instructions. As an example, the instructions in pseudo-codemight include the following.

-   -   High Level Instructions:    -   Run sample prep    -   Purify sample    -   Lysis operation    -   Run oncology screen test    -   Run sepsis test    -   Run lifestyle test    -   Send data to insurer    -   Read patient profile    -   Run sequencing test    -   Read gene type    -   Output genotype to cloud    -   Reassemble sequence    -   Medical_association(genotype)    -   Drug_association(genotype)    -   QuantifyDNA    -   Low Level Instructions:    -   Flow nucleotide (A, T, C, G)    -   Run PCR (temp1, period1, temp2, period2, temp3, period3, cycles)    -   Wash assay    -   Load bead (z) into well (x, y)    -   Read results for GSIC (x, y)    -   Synthesize primer (ATCCGGTTA)    -   Heat wells (54 deg C.)    -   Filter_data (filter type, parameters)    -   ADC (parameters)

The application may include or have access to a database of biologicalvalues such as genotypes, antibodies, SNPs, microbial species that canbe compared to the biological data output from an assay. Each data entryin the database may have one or more associated diagnostic, medical, ordrug parameters. Each data entry in the database may be associated witha set of instructions. The application receives data from the assay andcompares this to the database to determine the species or variant, finda recommended drug or therapy, or run the set of instructions. Thisexpedites the procedure from receiving data and providing a meaningfuloutput. For example, in a DNA sequencing assay where the data output isa stream of bases on the template nucleic acid, the application looks upeach base or partial sequence of bases in the database. Some of thesewill return no useful association for which the application doesnothing. Some of these bases will identify the species of the microbe inthe sample and the database may contain instructions to continuesequencing to determine the particular variant.

FIG. 7 illustrates schematically functional and structural features of afurther exemplary instrument comprising an application layer, anoperating system, and a set of primitives and resources. The Figureillustrates in particular that the operating system makes available tothe application(s) a set of assay processes including amplification,sample preparation, etc.

FIG. 8 is a flow diagram illustrating a process for analysing nucleicacid or fragments thereof. A various points in the flow, detectedoutputs are provided to inform workflow decisions, and workflow controldecisions are received. FIG. 9 illustrates schematically a cartridge 1suitable for performing this analysis. The cartridge may be configuredfor use with an instrument that provides fluids to the cartridge via afluid interface 2, and which exchanges data with the cartridge via adata interface 3. The cartridge comprises a controller 4 which controlsa fluid transport system 5 fluidly coupled to the fluid interface 2. Thecontroller also controls a set of reaction chambers 6, each of whichcomprises a set of sensors 7.

The invention claimed is:
 1. An apparatus for analysing nucleic acid andcomprising: a cartridge having a reaction chamber having a plurality ofwells located on the base of the chamber with respective sensors, thesensors configured or configurable to detect amplification, sequencing,and hybridisation in the reaction chambers; a fluid transport system forcontrolling the flow of fluids, including fluids containing said nucleicacid or fragments of said nucleic acid, to the reaction chambers tofacilitate a sequence of reactions including amplification, sequencing,and hybridisation of the nucleic acid and/or fragments thereof; and acontroller implemented on a computer, said computer coupled to saidsensors and to said fluid transport system, said sensors configured forobtaining detection results including intermediate reaction sequenceresults, said controller configured to provide a control signal to saidfluid transport system to dynamically direct the sequence of reactionsfor the analysis using the intermediate reaction sequence results,wherein the controller is programmed, based on data provided by thesensors, to determine an instance where there is no amplification, and,when the instance where there is no amplification is detected, thecontroller is programmed to provide a control signal to said fluidtransport system to stop any further reactions taking place, and whereinthe controller is further programmed to determine, based on dataprovided by the sensors, an instance where there is failure to detecthybridisation and provide a control signal to said computer to ignoredownstream sequencing reactions.
 2. The apparatus for analysing nucleicacid according to claim 1, wherein one or more of the sensors areconfigurable to detect two or more of amplification, sequencing, and/orhybridisation in the reaction chambers.
 3. The apparatus for analysingnucleic acid according to claim 1, wherein at least a part of said fluidtransport system is provided on the cartridge, and the cartridgecomprises fluid inlets and outlets for coupling the fluid transportsystem, or said part of said fluid transport system, to external fluidsources and fluid drains.
 4. The apparatus for analysing nucleic acidaccording to claim 1, wherein said sensors are all of the same sensortype.
 5. The apparatus for analysing nucleic acid according to claim 4,wherein said sensors are ChemFETs.
 6. The apparatus for analysingnucleic acid according to claim 1, wherein said fluid transport systemcomprises one or more fluid flow control gates, under the control ofsaid controller, for directing fluid to desired reaction chambers. 7.The apparatus for analysing nucleic acid according to claim 1, whereinthe reaction chamber comprises a plurality of identical sensors.
 8. Theapparatus for analysing nucleic acid according to claim 1, wherein thecontroller is configured for operating said reaction chambers andsensors; and wherein said controller is further configured to operatesaid reaction chambers and sensors in order to implement a workflowcomprising two or more of a genetic amplification, sequencing, andhybridisation, the workflow including one or more intermediate decisionpoints dependent upon result data obtained from said sensors.